User:J. Shaun Lott/BIOSCI 203



BIOSCI 203 Lab 2 - Protein structure
On the right-hand side of this page is a view of a protein structure shown using the Jmol viewer within Proteopedia. Jmol is an easy way to view protein structures. Use the left mouse button to rotate the protein model, the middle mouse button or scroll wheel to zoom in and out (option-click on a Mac), and the right mouse button for more options and information (control-click on a Mac). Try this out for yourself now ==>

The proteinstructure we will be looking at in this part of the lab is the N-terminal domain of the human 1-Cys peroxidase enzyme hORF6. Click here for an 'all atoms' view of the protein. This will show us all the atoms (except for the hydrogens) coloured by the 'CPK' colour scheme we talked about in lectures - blue for nitrogen atoms, red for oxygen atoms, grey for carbon atoms and yellow for sulfur atoms. Pretty hard to see what's going on, isn't it?

We can simplify things by just showing a cartoon that traces the path of the amino acid backbone; α-helices are shown as coils, and β-strands as arrows pointing in the direction of the C-terminus of the protein. Here is a version with the cartoon coloured from blue at the N-terminus to red at the C-terminus. Here is a version with the β-strands coloured yellow and the α-helices coloured pink.

It can help to see the backbone hydrogen bonds that define the secondary structure elements, and sometimes this is clearer shown as a backbone trace which just shows links between the Cα atoms, rather than a ribbon diagram. If we view the bonds in the protein in stick format, showing just the backbone atoms, we can easily see the backbone H-bond patterns that are distinctive for α-helices and β-sheets.

Q6: How many α-helices are there in this protein?

Q7: How many β-strands make up the β-sheet?

Q8: Is the topology of the β-sheet parallel, anti-parallel or mixed?

hORF6 is a member of the Peroxiredoxin (Prx) family of peroxidases, which are able to detoxify cytosolic hydroperoxides such as hydrogen peroxide (H2O2) by reducing them to water, with the help of a small molecule thiol-containing compound such as glutathione, represented here as RSH:

H+ + H2O2 + 2 RSH → H+ + 2 H2O + RSSR

hORF6 is an example of a 1-Cys Prx, and central to its enzymatic activity is its ability to maintain the active site cysteine residue in the charged (deprotonated) form of a thiolate ion (-S-) rather than the usual uncharged (protonated) sulfhydryl form (-SH).

Use the Henderson-Hasselbach equation to answer the questions below. (The pKa of the –SH group of free cysteine is 8.5. The cytosol of human cells is normally at pH 7.3. The pKa of the active site cysteine in a 1-Cys Prx enzyme has been measured at 6.0.)

Q9: What would be the % ionization of the –SH group of free cysteine in the cytosol?

Q10: What would be the % ionization of the –SH group of the Prx active site cysteine in the cytosol?



Now let’s look more closely at the hORF structure to see if we can identify what local features of the protein structure may influence the ability of the active site cysteine to ionize.

Here is a close-up view of the active site cysteine residue (Cys47) shown in 'ball and stick' representation. We want to know what other amino acid sidechains are close enough to influence the ionization of the cysteine sulfhydyl (-SH) group. If we now show other amino acid sidechains that have atoms within 4Å of the sulfur atom of Cys47, we can start to see what properties of these residues might be important in the ionization of Cys47.

'''Q11: Which residues have side chains close to Cys47? (Hint: try mousing over each of the displayed sidechains to identify them.)'''

Q12: What feature of the local environment around Cys47 might stabilize the existence of a thiolate ion?